MicroRNA-195-3p Promotes Hepatic Stellate Cell Activation And Liver Fibrosis By Suppressing PTEN Expression

Background: Hepatic fibrosis is a response in which the liver is continuously repairing damage after stimulation by external or intrinsic factors,and this response is characterized by abnormal accumulation of extracellular matrix（ECM）produced by cells after liver injury.When the injury persists,the injury repair in the liver is repeated,resulting in the continuous accumulation of extracellular matrix composed of type I collagen and other components in the liver,and the scar tissue that repairs and heals gradually thickens and gradually replaces the liver parenchyma,causing liver fibrosis to develop,which can eventually lead to cirrhosis and even liver cancer and other high mortality diseases.To address this mechanism,it is clear that activated hepatic stellate cells are the main source of extracellular matrix synthesis and secretion,and therefore it is important to study activated hepatic stellate cells.Recent studies have shown that activation of hepatic stellate cells is regulated by epigenetics,especially micro RNAs（miRNAs）,which have a significant impact on the activation and proliferation functions associated with hepatic stellate cells.Our experimental team found that miR-195-3p expression was significantly upregulated in HSC isolated from mice with carbon tetrachloride-induced liver fibrosis injury in a previous experimental study,and the upregulation was more pronounced with increasing liver fibrosis.In addition,in an in vitro transforming growth factor β（TGF-β1）-induced human immortalized hepatic stellate cell line（LX-2 cell）in vitro model,the This suggests that miR-195-3p may play a regulatory role in the development of liver fibrosis,but the specific regulatory mechanisms and regulatory functions are not yet clear.Objective: To investigate the possible regulatory role of miR-195-3p in liver fibrosis,and its downstream target genes.Methods: We first used miR-195-3p mimics in resting-state LX-2 cells in vitro to overexpress miR-195-3p.western blott,RT-q PCR,and Ed U were used to target the role of highly expressed miR-195-3p on LX-2 activation proliferation-related functions.Conversely,changes in cell activation and proliferation were similarly examined after knocking down miR-195-3p expression using miR-195-3p inhibitor in activated state LX-2 cells.After verifying the regulatory effect of miR-195-3p on LX-2 cell function,we investigated the specific regulatory mechanism by which miRNAs play an important regulatory role by targeting the 3’ UTR of downstream gene m RNAs.Therefore,we used the Target Scan website to predict the potential downstream target genes of miR-195-3p and to validate the predicted results.First,to confirm whether miR-195-3p binds to the predicted target gene definitively,we took a dual luciferase reporter assay,and second,we examined the expression of the predicted target gene in liver tissues of liver-injured mice and isolated hepatic stellate cells,as well as in LX-2cells after TGF-β1 stimulation.Immediately after that we transfected miR-195-3p mimics and overexpression plasmids of predicted target genes into LX-2 cells together using the same method,and on the other hand transfected miR-195-3p inhibitor into LX-2 cells together with small interfering RNA of predicted target genes.Further we investigated the downstream pathway of miR-195-3p target gene regulatory axis by querying the downstream regulatory pathway of the target gene through literature data,followed by transfection of miR-195-3p mimics/inhibitor in LX-2 cells to detect the changes in expression levels of genes related to the downstream regulatory pathway.Results: By comparing the results,it was found that overexpression of miR-195-3p promoted the proliferation of LX-2 cells,while the expression of fibrosis-related marker proteins α-SMA,Col1a1 and proliferation-related cyclin D1,C-myc were upregulated,indicating that LX-2 cells were transformed from the quiescent state to the activated state.In contrast,knockdown of miR-195-3p expression resulted in reduced LX-2 cell proliferation and decreased expression of fibrosis-related marker proteins and cell proliferation-related proteins,indicating a shift from an activated to a resting state.PTEN expression was down-regulated in liver tissue from liver-injured mice and in isolated hepatic stellate cells,and was also down-regulated in LX-2 cells after TGF-β1stimulation.After cotransfection of miR-195-3p mimics with overexpression plasmids of PTEN,it was observed that the regulatory effect of miR-195-3p high expression on LX-2 was partially suppressed by high expression of PTEN,and similarly,when miR-195-3p inhibitor was cotransfected with PTEN small interfering RNA（si RNA）,miR-195-3p ground expression produced a regulatory effect that was partially suppressed by PTEN si RNA.A review of the literature reveals that PTEN exerts a regulatory influence in the PI3K/Akt/mTOR signaling pathway.We detected significant upregulation of PI3K/Akt/mTOR regulatory pathway-related genes Akt/p-Akt and mTOR/p-mTOR protein levels in LX-2 cells transfected with miR-195-3p mimics,and conversely,PI3K/Akt/mTOR protein levels in LX-2 cells transfected with miRNA-195-3p inhibitor.Akt/mTOR signaling pathway-related protein expression was suppressed in LX-2 cells transfected with miRNA-195-3p inhibitor.Conclusion: The miR-195-3p/PTEN/PI3K/Akt/mTOR axis plays an important regulatory role in the activation and proliferation of hepatic stellate cells,inducing the expression of genes contributing to fibrosis,and has the potential to be used as a therapeutic target for liver fibrosis.